Role of the lectin pathway (LP) of complement in fighting two opportunistic pathogens, Candida albicans and Pseudomonas aeruginosa was assessed through a combination of in vitro assays supported by in vivo infection experiments using a unique mouse strain of total LP functional deficiency (MASP-2 -/- mouse). A highly significant difference in survival between MASP-2 -/- mice and MASP-2 +/+ littermates was observed following C. albicans infection with a lethal i.v. dose (1.4x106CFU/mouse), showing that MASP-2 -/- were significantly compromised. Challenging mice with a sub-lethal dose (4x105 CFU/mouse) revealed a significantly higher fungal load in kidneys, livers, lungs and spleens of MASP-2 -/- mice. IL-10 mRNA expression levels were only significantly upregulated in infected MASP-2 -/- mice, while mRNA expression of the protective cytokines IL-17 and INF-γ were only upregulated in MASP-2 +/+ mice. Challenging of MASP-2 -/- mice and MASP-2 +/+ controls with an intranasal dose (1x106 CFU/mouse) of P. aeruginosa revealed no significant difference in survival rates. The bacterial load with P. aeruginosa and mRNA expression profiles for TNF-α, IL-6, IL-10, MIP-2, IL-1β and INF-γ were similar in lungs of both mouse strains. The absence of the LP in MASP-2 -/- mice appears to make no difference in the susceptibility to P. aeruginosa infection as the alternative pathway seems to provide sufficient protection. The cDNA sequence of porcine MASP-2 was established to express an enzymatically inactive mutant form of porcine MASP-2 (pMASP-2A) in a mammalian cell line (CHOK1 cells). pMASP-2A was produced in large scale and used as an antigen to isolate recombinant inhibitory phage display antibodies. These antibodies will be analysed in porcine models of ischaemia/reperfusion (I/R) injury to assess the therapeutic potential of LP inhibition in limiting I/R injury and reduce morbidity and mortality in a clinically relevant experimental animal model of human disease.

Pseudomonas aeruginosa is an opportunistic pathogen and usually targets immunocompromised patients such as burn victims and patients with AIDS, cancer or cystic fibrosis (CF). Acquisition of this organism is associated with high mortality and can cause death within 24 hours. The main themes covered within this thesis are pathogenicity island characterisation in vitro and in vivo as well as investigation of in vivo virulence of P. aeruginosa. The rationale behind this focus is that 10-20% of the P. aeruginosa genome is variable between strains and large variable regions such as genomic (pathogenicity) islands are considered more likely to contribute to the differences in disease-causing ability between strains. The first project covers the development of a novel generic yeast-based genomic island capture method, which enables a complete genomic island to be present within a cloning vector. It was used in the characterisation of genomic islands in both P. aeruginosa and Escherichia coli. A novel genomic island in E. coli was captured and characterised. The second project investigates the contribution of two pathogenicity islands, PAPI- 1 and PAPI-2 to the in vivo virulence of P. aeruginosa PA14. Three pathogenicity island deletant isogenic mutants were tested for virulence in a murine acute respiratory model of infection developed for this project. The results showed that both pathogenicity islands contribute to virulence, but the presence of PAPI-2 is enough to maintain wild-type virulence. The third project covers the exploration of the role of quorum-sensing in the virulence of P. aeruginosa LES; one of the transmissible epidemic strains and the most common strain recovered from CF patients across the UK. The project assessed whether over-expression of quorum-sensing products is a reliable indicator of increased virulence within a murine acute respiratory model of infection. The results showed in general, over-expressing mutants were more virulent than deficient mutants, but there was one exception, LESB58.

No description available.

Prevalent worldwide, the protozoan parasite, Toxoplasma gondii, is an important cause of spontaneous abortion, ocular disease, mental retardation and encephalitis. Currently there are no human vaccines available. The first major aim of this study was to test potential HLA restricted peptide vaccines, previously shown to be protective in HLA-transgenic mice, against oocyst infection. The ability of entrapment within non-ionic surfactant vesicles to improve the efficacy of the HLA-B*0702 restricted vaccine was also studied. In parallel we tested the novel T. gondii ΔRPS13 live-attenuated vaccine against oocyst challenge. As determined by survival, only ΔRPS13 provided a measure of protection against oocyst challenge. We also demonstrated that the live vaccine induced a greater CD8+ T cell effector response than the adjuvanted peptide vaccine. Successful vaccination is in large part dependent on inducing an appropriate response in the primary host cell populations that consequently influences the development of adaptive immunity. Parasite induced macrophage arginase-1 expression, for example, has been shown to be influential during T. gondii infection. Arginase-1 expression is negatively regulated by Map Kinase Phosphatase-2 (MKP-2), the second major aim of the project was to study the effect of MKP-2 deficiency on T. gondii infection. MKP-2-/- mice were found to be more susceptible to infection with increased parasite growth and increased mortality compared with wild type mice. Increased susceptibility was associated with reduced serum nitrite levels and enhanced tissue arginase-1 expression although the Th1 response was unaltered. In vivo inhibition of iNOS and arginase-1 revealed that while NO production is of paramount importance in controlling parasite growth arginase-1 could also limit parasite growth independently. In vitro studies utilising macrophages confirmed a role for arginase-1 in parasite control. Results highlight a complex interaction between iNOS and arginase-1 and T. gondii in L-arginine metabolism but indicate that manipulation of early infection events influence disease outcome.

Acinetobacter is gaining importance as a pathogen in intensive care units (ICUs) due to the numerous outbreaks reported and the severity of the infections caused. Of particular interest in this project are neonates whose immune systems have not fully developed and affected by their prematurity, low birth weight, or various other conditions. The vulnerability of these neonates combined with the multidrug resistance of the clinical isolates of this organism poses a real threat to neonates. The understanding of its pathogenicity is still at an elementary stage despite the clinical evidence of nosocomial infections, and the concern of the FAOWHO (2006) regarding neonatal health due to the consumption of powdered infant formula (PIF). The ingestion of contaminated infant formula is a particularly unexplored mechanism of acquiring Acinetobacter infections. Therefore, this research work aimed to analyse the potential risk factors associated with the consumption of a contaminated infant formula administered enterally. The detection of the organism in PIF was examined. A selective chromogenic medium was designed, primarily for this purpose, and evaluated against other specific media. In addition, the desiccation persistence in infant formula was assessed. Biofilm formation inside nasogastric feeding tubes, survival in gastrointestinal fluids, and subsequent interactions with the host, as consequences of the ingestion of a feed containing Acinetobacter, were also investigated. Strains were finally screened for the presence of phospholipase genes. Based on the main findings, Acinetobacter showed the ability to persist over an extended storage period (2 years) in desiccated infant formula and recover after reconstitution. Bacterial cells in the contaminated formula formed biofilms and multiplied over time inside enteral feeding tubes causing all the subsequent fresh feeds to be also contaminated. When exposed to simulated gastrointestinal fluids, these cells did not show signs of complete viability loss except at an acidity level of pH 2.5 (lower than the normal neonatal stomach pH). Strains also successfully attached to colonic epithelial cells (Caco-2), and the majority were subsequently able invade the host cells. Twenty-four hour survival within the Caco-2 cells was also shown. Furthermore, Acinetobacter demonstrated the capacity to evade the bactericidal activity of macrophages, persist, and moreover multiply within these phagocytic cells. Genes encoding for the cytotoxic phospholipase enzymes were also detected in all the clinical isolates of this organism. In conclusion, Acinetobacter was established to possess multiple virulence factors that can potentially contribute to its pathogenicity once ingested.

The genus Yersinia consists of a heterogeneous collection of organisms, comprising the highly pathogenic species Yersinia pestis, enteropathogens Yersinia pseudotuberculosis and Yersinia enterocolitica as well as environmental species. The evolutionary history of Y. pestis has been well documented, but information on the evolutionary relationship between the other Yersiniae is less characterized. Y. enterocolitica is a diverse species classed into six different biotypes (BT), but only a single genome sequence for high-pathogenic BT 1B was available at the start of the project. This project looked into the dynamics shaping the pathogenic lineages in Yersinia. For each of the Y. enterocolitica BTs, a reference genome was sequenced, annotated and analysed in detail. Analysis of biochemical characteristics was carried out using a phenotypic microarray. A further 98 Y. enterocolitica, 36 Y. pseudotuberculosis/Y. pestis isolates and 81 environmental species were sequenced and analysed to investigate the evolutionary dynamics of the genus Yersinia. The Y. enterocolitica BTs form three distinct groups according to non-, low-, and high-pathogenic BT, each with a different set of accessory genes. The Y. enterocolitica core genome comprises ~3,300 CDSs. The non-pathogenic BT contains genes involved in environmental survival, whereas the high-pathogenic BT has a high-pathogenicity island and the unique Ysa type III secretion system. The low-pathogenic BTs share a second flagella cluster and an insect pathogenicity island. Apart from the virulence plasmid pYV, only 40 CDSs are shared in all pathogenic BTs including the adhesion invasion locus ail. The phenotypic microarray confirmed higher metabolic flexibility of the non-pathogenic BT, and decreased biochemical abilities in the low-pathogenic BTs, mirroring an increased number of pseudogenes. Looking at the whole genus, pathogenic lineages are visible at opposite ends of an evolutionary tree. Y. pestis has evolved out of Y. pseudotuberculosis, and both are distinct from Y. enterocolitica which developed out of the environmental Yersiniae. Fish pathogen Y. ruckeri forms a third independent pathogenic lineage. Despite the large evolutionary distance, comparing dynamics in Y. enterocolitica to Y. pestis it is clear that independent acquisition of the virulence plasmid and metabolic streamlining to specific hosts drove pathogen evolution. Whilst this has concurrently happened in Y. pestis, this development has been weighted differently in lineages of Y. enterocolitica leading to a high-pathogenic, human-adapted biotype and lowpathogenic biotypes, which can be adapted to human or animal hosts.

Campylobacter jejuni is the world’s major cause of gastroenteritis in humans. Although motility, toxin production, adhesion and invasion are some of the key factors associated with C. jejuni pathogenesis, their mechanism in the disease process remains unclear. The key aim of this project is to study the genetic basis of hyperinvasiveness in a group of six C. jejuni strains which have been reported as hyperinvasive into human intestinal cell lines. Here, genomotyping of the hyperinvasive C. jejuni was performed by comparative genomic hybridization (CGH) against four low invasive C. jejuni strains. A group of 67 genes were identified as being present or highly divergent/absent in the hyperinvasive versus low invasive C. jejuni strains. Of these, nine genes were present and six genes were highly divergent/absent in all hyperinvasive C. jejuni. The PCR screening of these 15 genes in nine additional low invasive C. jejuni strains showed a significant association with the hyperinvasive phenotype. The majority of identified genes encoded proteins with essential cellular and metabolic functions along with some genes with known virulence related roles. Thus, the hyperinvasive phenotype is characterised by different functional networks rather than a single gene or gene cluster. All strains showed an overall genetic variability and the capsule, lipooligosaccharide, flagellar biosynthesis and restriction modification regions were the most diverse. The hierarchical clustering based on comparative genomic hybridization (CGH) did not group together the hyperinvasive C. jejuni as a single group and these strains possessed different MLST profiles. The hyperinvasive C. jejuni strains were shown to contain additional genetic content by pooled suppressive subtractive hybridization (PSSH). Eleven inserts were identified in total which were variably distributed in the hyperinvasive C. jejuni strains. Of these four sequences were specific to the hyperinvasive C. jejuni as these were absent from all thirteen low invasive C. jejuni strains tested. The majority of sequences matched with genes in Campylobacter and other bacteria and one sequence had no homology with anything in the databases today. Since, there is no insert identified as present in all the hyperinvasive C. jejuni strains it can be suggested that each strain might have evolved a different mechanism for hyperinvasiveness and that this phenotype is a multifactorial process. C. jejuni 01/10 and 01/51 whole genome sequences identified no unique genetic content in either strain except for a prophage in C. jejuni 01/51. C. jejuni 01/10 was found to contain two prophages. C. jejuni 01/51 has a highly mosaic capsule locus with genes similar to C. jejuni subsp. doylei and C. lari capsular polysaccharide genes. Some genes with homology to the C. jejuni subsp. doylei capsule genes were also identified in C. jejuni 01/10 capsule region. This is evidence of genetic recombination with capsule genes from other pathogenic Campylobacter species which is not reported in the capsule region of other Campylobacter strains sequenced to date. This suggests that the highly diverse capsule in C. jejuni 01/10 and 01/51 is required for the hyperinvasive phenotype in these strains. This study has provided detailed insight into the genomic structure of the hyperinvasive C. jejuni strains and has highlighted genetic factors involved in their hyperinvasive phenotype.

There is a need for alternative “treatments” for killing microbes in wound and other body surfaces. Antimicrobial Photodynamic Therapy (APDT) and Electrochemically Activated Solutions (ECAS) are two recent developments in the field of biocide research that in the future may have widespread applications, replacing conventional antibiotics or disinfectants. The aims of the work described in this thesis are 5-fold. (1) Develop an in vitro model and determine the kill rates of bioluminescent species using bioluminescence light output (2) Measure the comparative effects of heavy water and reactive oxygen species (ROS) inhibitory agents against appropriate controls for both APDT and ECAS treatment and gain insight into general killing mechanisms (3) Develop methods to compare kill rates of target microbe (prokaryotic cells) against mammalian eukaryotic cells, by comparison of kill rates under the same treatment conditions and thus, assess the likelihood of cytotoxic damage to the host (4) Measure the potential of treatments to induce genotoxic damage to mammalian cells using a COMET assay (5) Measure the effects of treatments when the target is growing in biofilm mode (using a continuous matrix perfusion model) and compare standard treatments for fast and slow growing cells. A standard assay was developed containing target cell suspensions with killing agent ECAS or APDT (methylene blue (MB) combined with polychromatic light) and bioluminescence was measured using luminometer and viable count methods. For mechanistic study, the assay was repeated in the presence of ROS scavenger molecules. Fluorescence responses by probes singlet oxygen sensor green (SOSG), 3’-(p-aminophenyl) fluorescein (APF), 3’-(p-hydroxyphenyl) fluorescein (HPF) were measured using a fluorimeter to detect ROS in the presence or absence of specific ROS inhibitory agents. The potential cytotoxicity of APDT and ECAS against keratinocytes (H103) and lymphocytes (Jurkat cell) was measured using the neutral red test (for keratinocytes) and MTS assay (for lymphocytes). Trevigen’s comet assay kit was used to measure genotoxicity produced by ECAS and APDT towards lymphocytes, DNA damage was determined using epifluorescence microscopy. An in vitro flat-bed perfusion biofilm model was used to compare the effects of ECAS and APDT against biofilms, using a low light camera to measure bioluminescence within the biofilm. All data were compared using appropriate statistical analyses. The light output from the bioluminescent target species was highly proportional to the viable counts with high correlation (R2> 0.9). Order of killing susceptibility was S. aureus > E. coli > P. aeruginosa > MRSA. Kill rates measured using luminescent light output from lux-modified target species are more accurate than conventional viable count. The rapid assay method, coupled with the use of D2O, ROS-scavengers and fluorescent probes provided key insights into mechanisms of APDT and ECAS. It was confirmed that singlet oxygen is the main cytotoxic species for ADPT whilst a mixed system (hydroxyl radical plus singlet oxygen, and possibly other species) was involved for ECAS. APDT and ECAS are not particularly cytotoxic to mammalian cells (keratinocytes or lymphocytes); therefore a large safety margin of dose may exist to reduce the microbial cells without harm to mammalian cells. APDT is more genotoxic to lymphocytes than is ECAS but both are less toxic compared to H2O2 positive control. The in vitro flat-bed perfusion biofilm model was suitable to study both APDT and ECAS against biofilm cells. The results showed that biofilm was resistant compared to planktonic cells, and was able to recover easily post treatment. Slower growing cells take longer to recover following APDT.

The studies presented in this thesis describe the identification of novel capsid motifs involved in cell culture adaptation of FMDV A/Iran/87 A-. Field isolates of foot-and-mouth disease virus (FMDV) use integrins as receptors due to the presence of a conserved integrin-binding RGD motif located on the G-H loop of VP1, whereas cell-culture adapted variants can use other receptors such as heparan sulphate (HS). FMDV A/Iran/87 A- was isolated from a vaccine stock and has a major deletion within the VP1 G-H loop. This virus is unable to bind to integrins (as it lacks the RGD), and lacks the known HS contact residues. Sequence comparison identified a limited number of surface exposed residue changes, including a LEK to SAR tri-peptide at VP2 78-80 and a KE to EK di-peptide at VP2 130-131. Reverse genetics was used to investigate a functional role for these motifs in infection where it was found that VP2 80 (R) and 131 (K) were critical for infectivity. Mutagenesis was then used to introduce these motifs in to another type-A virus whilst simultaneously abrogating integrin-binding. Infectious virus could only be recovered when both the SAR and EK motifs were present suggesting that both the SAR and EK motifs are essential for infection, most likely by forming a novel receptor attachment site. Using immunofluorescence microscopy, cell surface-bound A/Iran/87 A- was rapidly internalised (within 5 mins), and co-localised with markers of early endosomes. This was characteristic of clathrin-mediated endocytosis and this conclusion was supported by observations that entry was inhibited by clathrin- and dynamin-inhibitors. However A/Iran/87 A- also co-localised with caveolin-1, a marker of caveolae, suggesting it may use more than one entry pathway. Taken together, this research may pave the way for future studies into rationally designed vaccine-viruses.

Translation of mRNA into protein represents the final step in the gene-expression pathway, driving the formation of the proteome from genomic information. The regulation of this process is a mechanism that is used to modulate gene expression in a wide range of biological situations. Protein synthesis is principally regulated at the initiation stage, allowing for rapid, reversible control of gene expression. Progress over recent years in determining the structures and activities of regulatory factors, and in mapping their interactions, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity, internal ribosome initiation and through sequence-specific RNA-binding proteins. This thesis focused on translational control during viral infection, where we investigated the role of Severe Acute Respiratory Syndrome non-structural protein 1 and Enterovirus 71 Internal Ribosome Entry Site in this process. To establish the function of SARS NSP1 protein in translation regulation we attempted the identification of NSP1 protein partners using several types of protein affinity chromatography. Using a wide range of approaches, we could not detect nor confirm the association of NSP1 with any cellular proteins. To dissect the role of FBP2, we engineered a wide range of recombinant FBP2 proteins of different lengths and analysed their interactions with IRES elements using biochemical techniques. This allowed us to characterize the interaction of EV71 IRES with initiation factors eIF4A, eIF4E, eIF4G and FBP2. Finally, we used chemical probing of RNA structure in solution to establish the secondary structure of the BiP IRES. We identified the formation of a structured RNA scaffold of 220 nucleotides comprising 3 major domains.